- Email: [email protected]
Proteolytic activity of Leuconostoc oenos
FEMS Microbiology Letters 150 (1997) 135^139
Proteolytic activity of Leuconostoc oenos. E¡ect on proteins and polypeptides from white wine M.C. Manca de Nadra a *, M.E. Far|èas a , M.V. Moreno-Arribas b , E. Pueyo b , M.C. Polo b ;
a
Facultad de Bioqu|èmica, Qu|èmica y Farmacia, Universidad Nacional de Tucumaèn and Centro de Referencias para Lactobacilos (CERELA), Chacabuco 145, 4000 Tucumaèn, Argentina b Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain Received 30 January 1997 ; revised 21 February 1997; accepted 1 March 1997
Abstract
The proteolytic activity of Leuconostoc oenos protease on white wine proteins and polypeptides was studied. Comparison of the peptide profiles before and after protease action demonstrated the presence of two new and an increase in two other peptide peaks. In total 56.7 mg l31 of peptides was liberated by action of the Lc.oenos proteases. Essential amino acids for Lc. oenos growth were liberated as free amino acids, and arginine, which has a stimulatory effect on Lc. oenos growth, was quantitatively the more important amino acid obtained by protease activity. Thus proteolytic activity serves to provide the cells with essential growth factors. Keywords : Leuconostoc oenos; Proteolytic activity ; White wine
1. Introduction
Leuconostoc oenos is generally the microorganism involved in malolactic fermentation during wine elaboration. Malolactic fermentation generally takes place in the wine after alcoholic fermentation when the medium is depleted of assimilable nitrogenous compounds [1]. Amino acids are important for the growth of Lc. oenos strains, both as nitrogen and carbon sources [2^5]. Feuillat et al. [6] found that
* Corresponding author. Tel.: (54) 81-310465; Fax: (54) 81-310465
the peptide fraction of molecular mass lower than 1000 Da supported growth of Lc. oenos strains. Wine proteins and peptides come from grape proteins and from hydrolysis through the proteolytic action of extracellular yeast's protease and from release of proteins and peptides during autolysis of the yeast [7]. Rollaèn et al. [8,9] reported, for the ¢rst time, the production of two extracellular proteases by each of four Lc. oenos strains isolated from wines and the partial characterization of the enzymes of Lc. oenos X2 L strain. The proteolytic system of Lc. oenos is important for two reasons. First, this organism has an absolute requirement for several amino acids [5]. Second, the
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 1 0 9 - 2
FEMSLE 7537 4-9-97
136
M.C. Manca de Nadra et al. / FEMS Microbiology Letters 150 (1997) 135^139
content of proteins and peptides in wine may cause wine turbidity and loss due to proteolytic activity reduces formation of a visible haze. Previous work on proteases from Lc. oenos was done by colorimetric determination of the amino acids liberated from grape juice, basal medium for Lc. oenos growth, albumin, gelatin and casein. The highest proteolytic activity was achieved with grape juice [8,9]. The aim of this work was to study the proteolytic e¡ect of Lc. oenos protease on wine proteins and polypeptides by determining the changes in the free and total amino acid content and in the peptide pro¢le. 2. Materials and methods
2.1. Microorganism Leuconostoc oenos X2 L
tinian wine [10].
was isolated from Argen-
2.2. Medium and culture conditions
The basal medium contained per l: 10 g yeast extract, 5.0 g glucose, 1.0 ml of Tween 80, and 170 ml of grape juice. Any alterations of these concentrations are noted in the text. The pH was adjusted to pH 4.5 with 0.1 M HCl before sterilization by autoclaving for 15 min at 121³C. 2.3. Determination of proteolytic activity
The assay mixtures for the di¡erent methods contained: cell-free culture supernatants (enzyme solution), substrate (white wine dialyzed and lyophilized) and 0.05 M citrate bu¡er, pH 5.0^1.0, ml of ¢nal volume. After 1 h incubation at 30³C, the reaction was stopped by the addition of 0.65 ml of 24% trichloroacetic acid (TCA). A control precipitated with TCA immediately before incubation was conducted in all cases. 2.3.1. Modi¢ed Cd-ninhydrin method
To a sample of TCA supernatant (20^100 Wl), depending on the concentration of amino acids expected, 1.7 ml of Cd-ninhydrin reagent was added [11].
2.3.2. o-Phthaldialdehyde (OPA) spectrophotometric assay
To a sample of TCA supernatant, 1 ml of OPA reagent was added [12]. 2.3.3. Method of Hull modi¢ed by Citti
To 0.65 ml of TCA supernatant 1.25 ml of carbonate-pyrophosphate bu¡er was added [13]. After 10 min of incubation at room temperature, 0.35 ml of Folin-Ciocalteau reagent was added and the blue color was read at 650 nm. 2.4. Isolation of the wine polypeptide and protein fraction
A Chardonnay white wine manufactured industrially from sul¢ted musts (80 mg l31 ), in 100 000 l tanks at 16^18³C, clari¢ed with 20 g bentonite/hl and 1 g gelatine/hl and tartrate stabilized, was used. To obtain macromolecular fraction, the wine was dialyzed against tap water using membranes with pore size of 3500 Daltons (Spectrum Medical Industries, Los Angeles, CA) for 48 h. The retentate was lyophilized. 2.5. Total protein
The proteins were determined by the reaction with Coomassie Brillant Blue G-250 [14]. 2.6. Analysis of free and total amino acids
The analysis was performed by the HPLC method of o-phthaldialdehyde derivatives, described in detail in Gonzalez del Llano et al. [15]. Total amino acids were determined after their hydrolysis at 110³C for 24 h, with 6 N HCl in vials sealed under vacuum in the same way as free amino acids. 2.7. Peptide analysis by HPLC
Analysis was performed by the method described by Moreno-Arribas et al. [16]. All separations were performed on a 150U3.9 mm i.d. Waters Nova-Pak C18 60 Aî, 4 Wm column. Eluent A was 0.1% tri£uoroacetic acid in water; eluent B was 0.1% tri£uoroacetic acid in acetonitrile. The gradient of B increased from 0 to 40% over 70 min. Flow rate
FEMSLE 7537 4-9-97
M.C. Manca de Nadra et al. / FEMS Microbiology Letters 150 (1997) 135^139
Fig. 1. HPLC analysis of peptides before (A) and after (B) of
Lc. oenos
137
X2 L protease action. With respect to the chromatograph before
the protease action, the numbers 14 and 15 indicate peaks without equivalent, numbers 2 and 9 indicate increased peaks, and the number 3 was not detected.
was 1 ml/min. Samples (50 through a 0.45
Wm
Wl)
previously ¢ltered
membrane ¢lter were injected.
L
nient methods (Cd-ninhydrin and OPA- -mercaptoethanol) that use reagents that react with
O
-amino
groups, providing increased sensitivity and direct determination of all products of proteolysis. To determine the activity of the enzyme on natural
3. Results and discussion
wine proteins as substrate, samples of a dialyzed and
Lc. oenos
X2 L was
lyophilized white wine were used. A comparative
obtained after 77 h incubation at 30³C in basal me-
study of the results from the three methods for pro-
è n et al. [8,9] utidium. In the previous paper Rolla
tease determination is presented in Table 1. The mi-
lized the methods of Hull modi¢ed by Citti et al. [13]
nor value of release of amino acids obtained by the
to evaluate proteolytic activity. This method relies
method of Hull modi¢ed by Citti et al. [13] can be
on the release of tyrosine- and tryptophan-contain-
explained by the speci¢ty of this reagent for the ami-
ing peptides that react with the Folin-Ciocalteau re-
no acids tyrosine and tryptophan. The OPA reagent
agent. These two amino acids are present in low
does not react with proline, the major amino acid of
concentrations in the macromolecular fraction of
wines and therefore the result obtained by this meth-
the wines and the method lacks sensitivity for deter-
od is also inferior to the result obtained by the nin-
mination of proteolytic activity in wines. For that
hydrin method. The speci¢c activity (determined by
reason we evaluated two more sensitive and conve-
Cd-ninhydrin method) on the wine lyophilized as
The exocellular protease of
Table 1 Proteolytic activity determination by di¡erent methods
W
Methods
Amino acids (nM)
Proteolytic activity ( mol/mg prot/h)
Cd-ninhydrin
0.16
4.09
OPA- -mercaptoethanol
L
0.14
3.30
Hull
0.08
1.60
Substrate : macromolecular fraction (MW
s
3500) of white wine.
The values are the mean of triplicate experiments.
FEMSLE 7537 4-9-97
138
M.C. Manca de Nadra et al. / FEMS Microbiology Letters 150 (1997) 135^139
Table 2 Free and total amino acids content before and after proteolytic activity of Lc. oenos Total amino acids (mg l31 ) Free amino acids (mg l31 ) After proteolytic activity (B) B 3A Control (A) After proteolytic activity (B) Control3 (A) Asp 31.4 33.4 2.0 Glu 22.9 34.1 11.2 Asn 16.2 17.4 1.2 Asx 51.6 62.0 Glx 100.1 118.3 Ser 31.0 32.3 1.3 24.3 28.6 Hys 25.7 27.2 1.5 25.9 30.6 Gly 18.5 19.8 1.3 29.6 35.4 Thr 29.2 32.0 2.8 27.8 33.5 Arg 39.8 43.0 3.2 36.2 42.9 Ala 52.8 55.1 2.3 44.3 50.7 Tyr 10.1 10.9 0.8 3.9 4.5 Met 8.1 9.1 1.0 3 3 Val 37.8 39.9 2.1 32.4 39.1 Trp 4.3 6.7 2.4 3 3 Phe 28.9 30.5 1.6 22.6 26.6 Ile 32.2 33.3 1.1 28.9 34.4 Leu 48.6 50.5 1.9 37.6 45.3 Lys 24.1 25.6 1.5 35.9 45.1 Total 461.6 500.8 39.2 501.1 597.0 3 See Section 2. Data are the mean of three replicates samples that are reproducible within þ 6%.
substrate, shows that 4.09 Wmol of amino acids was liberated. Taking into account the micromoles of amino acids liberates from 4 Wg of proteins (obtained as the di¡erence between the protein concentrations before and after protease action) 2.25% of the macromolecule was hydrolyzed. The e¡ect of protease action on release of individual amino acids from wine protein and polypeptides was evaluated by comparing samples taken before and after proteolysis (Table 2). The di¡erences between total and free amino acids after action of the protease is an indirect measure of peptides released from wine proteins. Then from the results of Table 2, 56.7 mg l31 of peptides has been released. That is also an important measure of amino acid availability because the peptides are more easily utilized by the cells than are amino acids. Amoroso et al. [5] reported that Lc. oenos X2 L required as growth factors L-asparagine, L-phenylalanine, L-histidine and methionine in synthetic medium and that these requirements increased when Lmalic and citric acids, two compounds normally present in wine, were added independently or com-
B3A
10.4 18.2 4.3 4.7 5.8 5.7 6.7 6.4 0.6
3
6.7
3
4.0 5.5 7.7 9.2
bined into the medium. Arginine, quantitatively the more important amino acid obtained by protease activity (3.2 mg l31 ), has a stimulatory e¡ect on growth of the microorganism. Fig. 1 shows the peptide pro¢les obtained by HPLC of the reaction mixture before (Fig. 1A) and after (Fig. 1B) incubation with Lc. oenos protease (Section 2). Several poorly resolved peaks corresponding to free amino acids [17] elute near the dead-volume. In the chromatogram corresponding to the control mixture before enzymatic treatment, 13 peptide peaks have been separated (Fig. 1A). Several changes can be observed in the peptide pro¢le after enzyme incubation (Fig. 1B). Two new peptide peaks named #14 and #15 are detected. The height of peaks #2 and #9 increase, and peptide #3 from Fig. 1A was not detected after enzyme incubation. These ¢ndings con¢rm the proteolytic activity of Lc. oenos on wine proteins. Feuillat et al. [6] found that the peptide fraction of molecular mass lower than 1000 Da supported growth of strains of Lb. plantarum, P. cerevisiae and Lc. oenos, while free amino acids only allowed growth of Lc. oenos. The proteo-
FEMSLE 7537 4-9-97
M.C. Manca de Nadra et al. / FEMS Microbiology Letters 150 (1997) 135^139 lytic system of
Lc. oenos
that liberates peptides and
essential amino acids is another important characteristic of growth necessary to conduct the malic acid decarboxylation.
[6] Feuillat,
M.,
Rochard,
J.
(1980)
Mise
en
ê t de levures au cours de la fermentation alcoolique du mou raisin. Conn. Vigne Vin 14, 37^52.
Microbiology
and
Biotechnology
(Fleet,
Ch.7, pp 225^242. Harwood Academic
G.H.,
Eds.),
Publishers, Switzer-
land. è n, G.C., Far| èas, M.E. and Manca de Nadra, M.C. (1993) [8] Rolla Protease
This work has been carried out within the framethe
and
[7] Charpentier, C. and Feuillat, M. (1993) Yeast autolysis. In :
Acknowledgments of
G.
evidence d'une production de proteases exocellulaires par les
Wine
work
Brillant,
139
Iberoamerican
Cooperation
Program
è n y Ciencia. from the Spanish Ministerio de Educacio The authors thank the ¢nancial support given by the Consejo de Investigaciones de la Universidad Nacio-
production
by
Leuconostoc oenos
strains
isolated
from wine. World J. Microbiol. Biotechnol. 9, 587^589. è n, G.C., Far| èas, M.E. and Manca de Nadra, M.C. (1995) [9] Rolla Characterization of two extracellular proteases from
nostoc oenos.
Leuco-
World J. Microbiol. Biotechnol. 11, 153^155.
[10] Strasser de Saad, A.M. and Manca de Nadra, M.C. (1987) Isolation and identi¢cation of the lactic acid bacteria from
è n (CIUNT) Argentina and the Comnal de Tucuma
Cafayate (Argentina) wines. Microbiol. Alim. Nutr. 5, 45^49.
è n Interministerial de Ciencia y Tecnolog| èa (Projisio
[11] Doi, E., Shibata, D. and Matoba, T. (1981) Modi¢ed colorimetric ninhydrin methods for peptidase assay. Anal. Biochem.
ect ALI-94-0737) Espan ì a.
118, 173^184. [12] Church, F.C., Swaisgood, H.E., Porter, D.H. and Catignani,
References
G. (1983) Spectrophotometric assay using o-phthaldialdehyde for
determination
of
proteolysis
in
milk
and
isolated
milk
proteins. J. Dairy Sci. 66, 1219^1227. [1] Guilloux-Benatier, M., Feuillat, M. and Ciol¢, B. (1985) Con-
[13] Citti, J.E., Sandine, W.E. and Elliker, P.R. (1963) Some ob-
é l'etude de la degradation de l'acid L-malic par les tri bution a
servations on the Hull method for measurement of proteolysis
èries lactiques isole è es du vin : e¡et stimulant des autolysats bacte
è ries lac[2] Bidan, P. (1967) Les facteurs de croissance des bacte
ment of the species of the genus
M.M.
(1976)
A
rapid
and
sensitive
method
for
quanti¢cation of microgram quantities of protein using the principle of protein dye-binding. Anal. Biochem. 72, 248^255.
tiques du vin. Ferment. Vini¢cation 1, 195^213. [3] Garvie, E.I. (1967) The growth factor and amino acid require-
conostoc paramesenteroides
in milk. J. Dairy Sci. 46, 337^342. [14] Bradford,
de levures. Vitis 24, 59^74.
Leuconostoc including LeuLeuconostoc oenos.
(sp nov) and
[15] Gonzalez de Llano, D., Polo, M.C., and Ramos, M. (1991) Production, isolation and identi¢cation of low molecular mass peptides from blue cheese by HPLC. J. Dairy Res. 58, 363^ 372.
J. Gen. Microbiol. 48, 439^447. [4] Kuensch, V., Temperli, A. and Mayer, K. (1974) Conversion
[16] Moreno-Arribas, M.V., Pueyo, E. and Polo, M.C. (1996) Pep-
of arginine to ornithine during malo-lactic fermentation in red
tides in musts and wines. Changes during the manufacture of cavas (sparkling wines). J. Agric. Food Chem. 44, 3783^3788.
Swiss wine. Am. J. Enol. Vitic. 25, 191^193. [5] Amoroso, M.J., Saguir, F.M. and Manca de Nadra, M.C.
[17] Acedo, M.I., Pueyo, E. and Polo, M.C. (1994) Preliminary
Leuconostoc
studies on peptides in wine by HPLC. Am. J. Enol. Vitic.
(1993) Variation of nutritional requirements of
oenos
by organic acids. J. Int. Sci. Vigne Vin, 27, 135^144.
45, 167^172.
FEMSLE 7537 4-9-97
Proteolytic activity of Leuconostoc oenos. E¡ect on proteins and polypeptides from white wine M.C. Manca de Nadra a *, M.E. Far|èas a , M.V. Moreno-Arribas b , E. Pueyo b , M.C. Polo b ;
a
Facultad de Bioqu|èmica, Qu|èmica y Farmacia, Universidad Nacional de Tucumaèn and Centro de Referencias para Lactobacilos (CERELA), Chacabuco 145, 4000 Tucumaèn, Argentina b Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain Received 30 January 1997 ; revised 21 February 1997; accepted 1 March 1997
Abstract
The proteolytic activity of Leuconostoc oenos protease on white wine proteins and polypeptides was studied. Comparison of the peptide profiles before and after protease action demonstrated the presence of two new and an increase in two other peptide peaks. In total 56.7 mg l31 of peptides was liberated by action of the Lc.oenos proteases. Essential amino acids for Lc. oenos growth were liberated as free amino acids, and arginine, which has a stimulatory effect on Lc. oenos growth, was quantitatively the more important amino acid obtained by protease activity. Thus proteolytic activity serves to provide the cells with essential growth factors. Keywords : Leuconostoc oenos; Proteolytic activity ; White wine
1. Introduction
Leuconostoc oenos is generally the microorganism involved in malolactic fermentation during wine elaboration. Malolactic fermentation generally takes place in the wine after alcoholic fermentation when the medium is depleted of assimilable nitrogenous compounds [1]. Amino acids are important for the growth of Lc. oenos strains, both as nitrogen and carbon sources [2^5]. Feuillat et al. [6] found that
* Corresponding author. Tel.: (54) 81-310465; Fax: (54) 81-310465
the peptide fraction of molecular mass lower than 1000 Da supported growth of Lc. oenos strains. Wine proteins and peptides come from grape proteins and from hydrolysis through the proteolytic action of extracellular yeast's protease and from release of proteins and peptides during autolysis of the yeast [7]. Rollaèn et al. [8,9] reported, for the ¢rst time, the production of two extracellular proteases by each of four Lc. oenos strains isolated from wines and the partial characterization of the enzymes of Lc. oenos X2 L strain. The proteolytic system of Lc. oenos is important for two reasons. First, this organism has an absolute requirement for several amino acids [5]. Second, the
0378-1097 / 97 / $17.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 7 ) 0 0 1 0 9 - 2
FEMSLE 7537 4-9-97
136
M.C. Manca de Nadra et al. / FEMS Microbiology Letters 150 (1997) 135^139
content of proteins and peptides in wine may cause wine turbidity and loss due to proteolytic activity reduces formation of a visible haze. Previous work on proteases from Lc. oenos was done by colorimetric determination of the amino acids liberated from grape juice, basal medium for Lc. oenos growth, albumin, gelatin and casein. The highest proteolytic activity was achieved with grape juice [8,9]. The aim of this work was to study the proteolytic e¡ect of Lc. oenos protease on wine proteins and polypeptides by determining the changes in the free and total amino acid content and in the peptide pro¢le. 2. Materials and methods
2.1. Microorganism Leuconostoc oenos X2 L
tinian wine [10].
was isolated from Argen-
2.2. Medium and culture conditions
The basal medium contained per l: 10 g yeast extract, 5.0 g glucose, 1.0 ml of Tween 80, and 170 ml of grape juice. Any alterations of these concentrations are noted in the text. The pH was adjusted to pH 4.5 with 0.1 M HCl before sterilization by autoclaving for 15 min at 121³C. 2.3. Determination of proteolytic activity
The assay mixtures for the di¡erent methods contained: cell-free culture supernatants (enzyme solution), substrate (white wine dialyzed and lyophilized) and 0.05 M citrate bu¡er, pH 5.0^1.0, ml of ¢nal volume. After 1 h incubation at 30³C, the reaction was stopped by the addition of 0.65 ml of 24% trichloroacetic acid (TCA). A control precipitated with TCA immediately before incubation was conducted in all cases. 2.3.1. Modi¢ed Cd-ninhydrin method
To a sample of TCA supernatant (20^100 Wl), depending on the concentration of amino acids expected, 1.7 ml of Cd-ninhydrin reagent was added [11].
2.3.2. o-Phthaldialdehyde (OPA) spectrophotometric assay
To a sample of TCA supernatant, 1 ml of OPA reagent was added [12]. 2.3.3. Method of Hull modi¢ed by Citti
To 0.65 ml of TCA supernatant 1.25 ml of carbonate-pyrophosphate bu¡er was added [13]. After 10 min of incubation at room temperature, 0.35 ml of Folin-Ciocalteau reagent was added and the blue color was read at 650 nm. 2.4. Isolation of the wine polypeptide and protein fraction
A Chardonnay white wine manufactured industrially from sul¢ted musts (80 mg l31 ), in 100 000 l tanks at 16^18³C, clari¢ed with 20 g bentonite/hl and 1 g gelatine/hl and tartrate stabilized, was used. To obtain macromolecular fraction, the wine was dialyzed against tap water using membranes with pore size of 3500 Daltons (Spectrum Medical Industries, Los Angeles, CA) for 48 h. The retentate was lyophilized. 2.5. Total protein
The proteins were determined by the reaction with Coomassie Brillant Blue G-250 [14]. 2.6. Analysis of free and total amino acids
The analysis was performed by the HPLC method of o-phthaldialdehyde derivatives, described in detail in Gonzalez del Llano et al. [15]. Total amino acids were determined after their hydrolysis at 110³C for 24 h, with 6 N HCl in vials sealed under vacuum in the same way as free amino acids. 2.7. Peptide analysis by HPLC
Analysis was performed by the method described by Moreno-Arribas et al. [16]. All separations were performed on a 150U3.9 mm i.d. Waters Nova-Pak C18 60 Aî, 4 Wm column. Eluent A was 0.1% tri£uoroacetic acid in water; eluent B was 0.1% tri£uoroacetic acid in acetonitrile. The gradient of B increased from 0 to 40% over 70 min. Flow rate
FEMSLE 7537 4-9-97
M.C. Manca de Nadra et al. / FEMS Microbiology Letters 150 (1997) 135^139
Fig. 1. HPLC analysis of peptides before (A) and after (B) of
Lc. oenos
137
X2 L protease action. With respect to the chromatograph before
the protease action, the numbers 14 and 15 indicate peaks without equivalent, numbers 2 and 9 indicate increased peaks, and the number 3 was not detected.
was 1 ml/min. Samples (50 through a 0.45
Wm
Wl)
previously ¢ltered
membrane ¢lter were injected.
L
nient methods (Cd-ninhydrin and OPA- -mercaptoethanol) that use reagents that react with
O
-amino
groups, providing increased sensitivity and direct determination of all products of proteolysis. To determine the activity of the enzyme on natural
3. Results and discussion
wine proteins as substrate, samples of a dialyzed and
Lc. oenos
X2 L was
lyophilized white wine were used. A comparative
obtained after 77 h incubation at 30³C in basal me-
study of the results from the three methods for pro-
è n et al. [8,9] utidium. In the previous paper Rolla
tease determination is presented in Table 1. The mi-
lized the methods of Hull modi¢ed by Citti et al. [13]
nor value of release of amino acids obtained by the
to evaluate proteolytic activity. This method relies
method of Hull modi¢ed by Citti et al. [13] can be
on the release of tyrosine- and tryptophan-contain-
explained by the speci¢ty of this reagent for the ami-
ing peptides that react with the Folin-Ciocalteau re-
no acids tyrosine and tryptophan. The OPA reagent
agent. These two amino acids are present in low
does not react with proline, the major amino acid of
concentrations in the macromolecular fraction of
wines and therefore the result obtained by this meth-
the wines and the method lacks sensitivity for deter-
od is also inferior to the result obtained by the nin-
mination of proteolytic activity in wines. For that
hydrin method. The speci¢c activity (determined by
reason we evaluated two more sensitive and conve-
Cd-ninhydrin method) on the wine lyophilized as
The exocellular protease of
Table 1 Proteolytic activity determination by di¡erent methods
W
Methods
Amino acids (nM)
Proteolytic activity ( mol/mg prot/h)
Cd-ninhydrin
0.16
4.09
OPA- -mercaptoethanol
L
0.14
3.30
Hull
0.08
1.60
Substrate : macromolecular fraction (MW
s
3500) of white wine.
The values are the mean of triplicate experiments.
FEMSLE 7537 4-9-97
138
M.C. Manca de Nadra et al. / FEMS Microbiology Letters 150 (1997) 135^139
Table 2 Free and total amino acids content before and after proteolytic activity of Lc. oenos Total amino acids (mg l31 ) Free amino acids (mg l31 ) After proteolytic activity (B) B 3A Control (A) After proteolytic activity (B) Control3 (A) Asp 31.4 33.4 2.0 Glu 22.9 34.1 11.2 Asn 16.2 17.4 1.2 Asx 51.6 62.0 Glx 100.1 118.3 Ser 31.0 32.3 1.3 24.3 28.6 Hys 25.7 27.2 1.5 25.9 30.6 Gly 18.5 19.8 1.3 29.6 35.4 Thr 29.2 32.0 2.8 27.8 33.5 Arg 39.8 43.0 3.2 36.2 42.9 Ala 52.8 55.1 2.3 44.3 50.7 Tyr 10.1 10.9 0.8 3.9 4.5 Met 8.1 9.1 1.0 3 3 Val 37.8 39.9 2.1 32.4 39.1 Trp 4.3 6.7 2.4 3 3 Phe 28.9 30.5 1.6 22.6 26.6 Ile 32.2 33.3 1.1 28.9 34.4 Leu 48.6 50.5 1.9 37.6 45.3 Lys 24.1 25.6 1.5 35.9 45.1 Total 461.6 500.8 39.2 501.1 597.0 3 See Section 2. Data are the mean of three replicates samples that are reproducible within þ 6%.
substrate, shows that 4.09 Wmol of amino acids was liberated. Taking into account the micromoles of amino acids liberates from 4 Wg of proteins (obtained as the di¡erence between the protein concentrations before and after protease action) 2.25% of the macromolecule was hydrolyzed. The e¡ect of protease action on release of individual amino acids from wine protein and polypeptides was evaluated by comparing samples taken before and after proteolysis (Table 2). The di¡erences between total and free amino acids after action of the protease is an indirect measure of peptides released from wine proteins. Then from the results of Table 2, 56.7 mg l31 of peptides has been released. That is also an important measure of amino acid availability because the peptides are more easily utilized by the cells than are amino acids. Amoroso et al. [5] reported that Lc. oenos X2 L required as growth factors L-asparagine, L-phenylalanine, L-histidine and methionine in synthetic medium and that these requirements increased when Lmalic and citric acids, two compounds normally present in wine, were added independently or com-
B3A
10.4 18.2 4.3 4.7 5.8 5.7 6.7 6.4 0.6
3
6.7
3
4.0 5.5 7.7 9.2
bined into the medium. Arginine, quantitatively the more important amino acid obtained by protease activity (3.2 mg l31 ), has a stimulatory e¡ect on growth of the microorganism. Fig. 1 shows the peptide pro¢les obtained by HPLC of the reaction mixture before (Fig. 1A) and after (Fig. 1B) incubation with Lc. oenos protease (Section 2). Several poorly resolved peaks corresponding to free amino acids [17] elute near the dead-volume. In the chromatogram corresponding to the control mixture before enzymatic treatment, 13 peptide peaks have been separated (Fig. 1A). Several changes can be observed in the peptide pro¢le after enzyme incubation (Fig. 1B). Two new peptide peaks named #14 and #15 are detected. The height of peaks #2 and #9 increase, and peptide #3 from Fig. 1A was not detected after enzyme incubation. These ¢ndings con¢rm the proteolytic activity of Lc. oenos on wine proteins. Feuillat et al. [6] found that the peptide fraction of molecular mass lower than 1000 Da supported growth of strains of Lb. plantarum, P. cerevisiae and Lc. oenos, while free amino acids only allowed growth of Lc. oenos. The proteo-
FEMSLE 7537 4-9-97
M.C. Manca de Nadra et al. / FEMS Microbiology Letters 150 (1997) 135^139 lytic system of
Lc. oenos
that liberates peptides and
essential amino acids is another important characteristic of growth necessary to conduct the malic acid decarboxylation.
[6] Feuillat,
M.,
Rochard,
J.
(1980)
Mise
en
ê t de levures au cours de la fermentation alcoolique du mou raisin. Conn. Vigne Vin 14, 37^52.
Microbiology
and
Biotechnology
(Fleet,
Ch.7, pp 225^242. Harwood Academic
G.H.,
Eds.),
Publishers, Switzer-
land. è n, G.C., Far| èas, M.E. and Manca de Nadra, M.C. (1993) [8] Rolla Protease
This work has been carried out within the framethe
and
[7] Charpentier, C. and Feuillat, M. (1993) Yeast autolysis. In :
Acknowledgments of
G.
evidence d'une production de proteases exocellulaires par les
Wine
work
Brillant,
139
Iberoamerican
Cooperation
Program
è n y Ciencia. from the Spanish Ministerio de Educacio The authors thank the ¢nancial support given by the Consejo de Investigaciones de la Universidad Nacio-
production
by
Leuconostoc oenos
strains
isolated
from wine. World J. Microbiol. Biotechnol. 9, 587^589. è n, G.C., Far| èas, M.E. and Manca de Nadra, M.C. (1995) [9] Rolla Characterization of two extracellular proteases from
nostoc oenos.
Leuco-
World J. Microbiol. Biotechnol. 11, 153^155.
[10] Strasser de Saad, A.M. and Manca de Nadra, M.C. (1987) Isolation and identi¢cation of the lactic acid bacteria from
è n (CIUNT) Argentina and the Comnal de Tucuma
Cafayate (Argentina) wines. Microbiol. Alim. Nutr. 5, 45^49.
è n Interministerial de Ciencia y Tecnolog| èa (Projisio
[11] Doi, E., Shibata, D. and Matoba, T. (1981) Modi¢ed colorimetric ninhydrin methods for peptidase assay. Anal. Biochem.
ect ALI-94-0737) Espan ì a.
118, 173^184. [12] Church, F.C., Swaisgood, H.E., Porter, D.H. and Catignani,
References
G. (1983) Spectrophotometric assay using o-phthaldialdehyde for
determination
of
proteolysis
in
milk
and
isolated
milk
proteins. J. Dairy Sci. 66, 1219^1227. [1] Guilloux-Benatier, M., Feuillat, M. and Ciol¢, B. (1985) Con-
[13] Citti, J.E., Sandine, W.E. and Elliker, P.R. (1963) Some ob-
é l'etude de la degradation de l'acid L-malic par les tri bution a
servations on the Hull method for measurement of proteolysis
èries lactiques isole è es du vin : e¡et stimulant des autolysats bacte
è ries lac[2] Bidan, P. (1967) Les facteurs de croissance des bacte
ment of the species of the genus
M.M.
(1976)
A
rapid
and
sensitive
method
for
quanti¢cation of microgram quantities of protein using the principle of protein dye-binding. Anal. Biochem. 72, 248^255.
tiques du vin. Ferment. Vini¢cation 1, 195^213. [3] Garvie, E.I. (1967) The growth factor and amino acid require-
conostoc paramesenteroides
in milk. J. Dairy Sci. 46, 337^342. [14] Bradford,
de levures. Vitis 24, 59^74.
Leuconostoc including LeuLeuconostoc oenos.
(sp nov) and
[15] Gonzalez de Llano, D., Polo, M.C., and Ramos, M. (1991) Production, isolation and identi¢cation of low molecular mass peptides from blue cheese by HPLC. J. Dairy Res. 58, 363^ 372.
J. Gen. Microbiol. 48, 439^447. [4] Kuensch, V., Temperli, A. and Mayer, K. (1974) Conversion
[16] Moreno-Arribas, M.V., Pueyo, E. and Polo, M.C. (1996) Pep-
of arginine to ornithine during malo-lactic fermentation in red
tides in musts and wines. Changes during the manufacture of cavas (sparkling wines). J. Agric. Food Chem. 44, 3783^3788.
Swiss wine. Am. J. Enol. Vitic. 25, 191^193. [5] Amoroso, M.J., Saguir, F.M. and Manca de Nadra, M.C.
[17] Acedo, M.I., Pueyo, E. and Polo, M.C. (1994) Preliminary
Leuconostoc
studies on peptides in wine by HPLC. Am. J. Enol. Vitic.
(1993) Variation of nutritional requirements of
oenos
by organic acids. J. Int. Sci. Vigne Vin, 27, 135^144.
45, 167^172.
FEMSLE 7537 4-9-97